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Received Jan 16, 2017; Revised May 10, 2017; Accepted May 23, 2017
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1. Introduction
1.1. Background
Aptamers are single-stranded nucleic acid sequences capable of specific high-affinity binding [1–4]. This makes them attractive candidates as recognition molecules in diagnostics and therapeutics. Aptamers are isolated by systematic evolution of ligands by conventional exponential enrichment (SELEX), which involves several iterative steps of incubation with target, washing away of weak binders, and PCR amplification of strong binders.
Aptamer selection is complex. Many variables such as library size, quantity of target, temperature, selection buffer, pH, degree of PCR amplification, and use of mutation or recombination diversification need to be considered. Due in part to these factors, less than 30% of classical SELEX experiments are successful in isolating aptamers with dissociation constants less than 30 nM [5]. Understanding the dynamics of the selection process is extremely important; but what does this entail? The DNA required to fully represent the number of permutations in a 75-base aptamer library would be roughly equal to the mass of the moon [6]. In order to represent this immense sequence space, an initial SELEX library may contain up to 1015 molecules. A rigorous empirical analysis of anything close to this number of library members is simply not feasible.
Nevertheless, empirical analyses of smaller fractions of a DNA aptamer libraries have been undertaken. The two main empirical selection analysis techniques used are high density DNA microarrays and high-throughput sequencing (HTS). Briefly, high density microarrays can contain up to approximately 1 million features, each representing an aptamer in a library. After array incubation with fluorescent target and a washing step, the binding affinity score of all aptamers on the array can be measured by fluorescence scanning. Platt et al. [7], Knight et al. [8], and Rowe et al. [9] used microarrays to both evolve aptamers and gain insight into an aptamer binding landscape. Additionally, DNA microarray data has been applied to aptamer specificity landscapes [10], fitness landscape morphology [11], and aptamer affinity maturation [12]. In comparison, the possible sequence space coverage using HTS is much greater, with Illumina’s...